In the SV40 lytic infection of African green monkey kidney cells, SV40 agnogene, a sequence within the proximal late genome region, is expressed. A 7900 Mr (61 amino acid) polypeptide (the agnoprotein) was detected. The identity of this protein has been confirmed by comparing its amino-terminal sequence with that predicted from the SV40 nucleotide sequence. The agnoprotein accumulated late in the lytic cycle and had a half-life of approximately 2 hours. The highly basic nature of the protein and its affinity for both single- and doublestranded DNA suggested a possible regulatory role of this protein in nucleoprotein interactions.